Electronic Supplementary Material (ESI) for Analytical Methods.
This journal is © The Royal Society of Chemistry 2015
Electronic Supplementary Information
A rapid genomic DNA extraction method and its combination with helicase dependent
amplification for the detection of genetically modified maize
Eric Gonzalez Garcia1, Andreas H. Farnleitner2, Robert L. Mach2, Rudolf Krska3, Kurt Brunner1
1
Vienna University of Technology – IFA-Tulln, Center for Analytical Chemistry, Konrad
Lorenz Str. 20, 3430, Tulln, Austria
2
Vienna University of Technology, Institute of Chemical Engineering, Gumperdorfer Str. 1a,
1060, Vienna, Austria
3
University of Natural Resources and Life Sciences, Department IFA-Tulln, Konrad Lorenz Str.
20, 3430, Tulln, Austria
1.
2.
Materials and methods ..............................................................................................................2
1.1 Extraction procedures......................................................................................................2
CTAB extraction protocol ...............................................................................................2
Wizard® Genomic DNA purification kit .........................................................................2
Results.......................................................................................................................................2
2.1 Optimization of incubation times with proteinase K.......................................................2
2.2 Optimization of the best temperature of extraction.........................................................3
2.3 Addition of chemicals to the sodium phosphate buffer...................................................4
2.4 Pretreatment of samples with solvents ............................................................................4
1
1. Materials and Methods
1.1
Extraction procedures
CTAB extraction protocol
The extraction was carried out based on the maize DNA isolation method proposed by the
Community Reference Laboratory for GM Food and Feed, 2007. Briefly, genomic DNA was
extracted by performing a thermal lysis (65oC, 1h) of the sample with the extraction buffer (1.4
M NaCl, 2% (w/v) cetyltrimethylammonium bromide (CTAB), 0.1M Tris-Base (pH=8), 0.02M
EDTA (pH=8) and 1% polyvinyl pyrrolidone 40000). DNA was later precipitated with the
precipitation buffer (1% (w/v) CTAB, 0.05 M Tris (pH=8), 0.01 M EDTA (pH=8)) and washed
with 70% ethanol. Resuspension of the DNA was done afterwards in 10 mM Tris buffer at 65oC
for 20 min and stored at -20oC until further use. All reagents were from Roth (Karlsruhe,
Germany). Each extraction was done in triplicates.
Wizard® Genomic DNA Purification Kit
The protocol was carried out according to the manufacturer. Briefly, 40mg of ground maize
powder were mixed with the nuclei lysis solution and incubated at 65oC for 15 min. RNase
solution was afterwards added and further incubated at 37oC for 15 min. The protein
precipitation solution was then added and the mixture was centrifuged at 15000 g. The
supernatant was later removed and mixed with room temperature isopropanol. After
centrifugation, the pellet formed was washed with 70% ethanol and air dried. The genomic DNA
extracted was rehydrated with DNA Rehydration Solution and incubated at 65oC for 1 h.
Solutions were then stored at -20oC until further use. Each extraction was done in triplicates.
2. Results
2.1
Optimization of incubations times with proteinase K
24.00
Cq
23.50
23.00
22.50
22.00
21.50
10
30
Time (min)
45
60
Figure 1. Optimization of incubation times when digesting the samples with proteinase K.
2
2.2
Optimization of the best temperature of extraction
25.00
24.50
Cq
24.00
23.50
23.00
22.50
22.00
50
55
60
65
70
Temperature (°C)
75
Figure 2. Screening for the best temperature of extraction.
3
80
85
90
PB
S
PB + p
S + ro
PB Na tein
S + Cl as
PB Na (10 e K
S + Cl 0m
(1 M
PB NaC 50m )
S + l(2 M
PB KC 00m )
S + l(1 M
PB KC 00m )
PB S l(
S + 1 M
PB + E KC 50m )
S + DT l(2 M
A 00 )
PB EDT (2 mM
S + A mm )
(1
PB Na2 0 m ol/l
S + SO m )
o
PB GuH 3 (0 l/l)
S + C .65
PB Gu l (0. %)
S + H 05
G Cl %
PB uH (0.1 )
S + Cl %
PB Gu (0.5 )
PB S + HC %
S+ G l( )
PB Tw uHC 1 %
S
e l )
PB + T en (3 %
S + we 20 )
e
PB Tw n 2 (1%
S + een 0 ( )
PB Tw 20 5%
)
S
e
PB + T en (10%
S + we 80 )
Tw en (1%
e 80 )
PB en 8 (5%
S+ 0
)
SD (10%
PB S
( )
PB S + 0.5%
S
S+ D )
SD S(1%
S( )
1.
5%
)
Cq
2.3
2.4
Addition of chemicals to the sodium phosphate buffer
36.00
34.00
32.00
30.00
28.00
26.00
24.00
22.00
20.00
Buffers
Figure 3. Influence of the usage of different chemicals in the sodium phosphate buffer in the Cq
value of the extracts.
Pretreatment of samples with solvents
Table 1. Results from a pretreatment of the maize powder samples with solvents.
Cq ± STD
Sample
CH3Cl
CH3Cl-EtoAc (1:1)
EtoAc
23.87 ± 0.68
24.22 ± 0.86
24.82 ± 0.99
4